Human food and animal feed derived from many grains are deficient in the sulfur amino acids, methionine and cysteine, which are required in the animal diet. In corn, the sulfur amino acids are the third most limiting amino acids, after lysine and tryptophan, for the dietary requirements of many animals. The use of soybean meal, which is rich in lysine and tryptophan, to supplement corn in animal feed diets is limited by the low sulfur amino acid content of the legume. As a result, synthetic sources of methionine need to be added to provide nutritional value to feed rations. Thus, an increase in the sulfur amino acid content of either corn or soybean would improve the nutritional quality of these mixtures and reduce the need for further supplementation through addition of more expensive methionine. Similarly the use of rapeseed (Brassica napus) meal as animal feed ingredient would be enhanced by increasing the sulfur amino acid content of the seeds.
The amino acid content of seeds is determined primarily by the storage proteins which are synthesized during seed development and which serve as a major nutrient reserve following germination. The quantity of protein in seeds varies from about 10% of the dry weight in cereals to 20-40% of the dry weight of legumes. In many seeds the storage proteins account for 50% or more of the total protein.
Efforts to improve the sulfur amino acid content of crops through plant breeding have met with limited success on the laboratory scale and no success commercially. A mutant corn line which had an elevated whole-kernel methionine concentration was isolated from corn cells grown in culture by selecting for growth in the presence of inhibitory concentrations of lysine plus threonine (Phillips et al., Cereal Chem., 62:213-218 (1985)). However, agronomically-acceptable cultivars have not yet been derived and commercialized from this line.
Because of their abundance plant seed storage proteins were among the first proteins to be isolated. Only recently, however, have the amino acid sequences of some of these proteins been determined with the use of molecular genetic techniques. These techniques have also provided information about the genetic signals that control the seed-specific expression and the intracellular targeting of these proteins.
A number of sulfur-rich plant seed storage proteins have been identified and their corresponding genes have been isolated. A gene in corn for a 15 kD zein protein containing 11% methionine and 5% cysteine (Pedersen et al., J. Biol. Chem., 261:6279-6284 (1986)) and a gene for a 10 kD zein protein containing 22% methionine and 3% cysteine have been isolated (Kirihara et al., Mol. Gen. Genet., 21:477-484 (1988); Kirihara et al., Gene, 71:359-370 (1988)). From rice a gene coding for a 10 kD seed prolamin containing 19% methionine and 10% cysteine has been isolated (Masumura et al., Plant Mol. Biol., 12:123-130 (1989)). A high sulfur zein (HSZ) seed storage protein, related but distinct in structure to the 10 kD zein protein, was described by Chui et at. ((1992) WO92/14822).
There have been several reports on the expression of seed storage protein genes in transgenic plants. The high sulfur 2S albumin from Brazil nut protein has been expressed in rapeseed (Brassica napus) seeds at a level that resulted in an up to 33% increase in the level of methionine of the salt-extractable seed protein fraction (Altenbach et al., Plant Mol. Biol., 18:235-245 (1992)).
To date, there are no reports of the transfer of the monocotylodonous 10 kD zein gene into and its expression in dicotyledonous plant species such as tobacco, Brassica napus, soybean, sunflower, Arabidopsis, etc.
In order to increase the sulfur amino acid content of seeds it would be useful to develop a method to express a gene coding for a seed storage protein that is rich in the sulfur-containing amino acid, methionine, resulting in high levels of methionine in the seeds of transformed plants. It would be desirable for the storage protein to be compatible with those of the target crop plant and thus have no detrimental effect on seed development. Furthermore, it would be desirable that the protein come from a source that is generally regarded as safe for human food and animal feed. Crop plants obtained by this method would be valuable as sources of sulfur amino acid enriched meal.